During neural development, axons are guided to their final destination by a large number of molecular cues-attractive and repulsive signals that instruct the cytoskeleton to redirect the direction of growth. One of these signals involves the interaction of Semaphorins with Plexin. The presence of this signal is conveyed to the cytoskeleton by another molecule, MICAL (Molecule Interacting with CasL), a multidomain protein with an FAD-containing hydroxylase (MICALfd ) domain. We have determined the structure of MICALfd and showed that it catalyzes the reduction of O2 to H20 2 using NADPH. This activity is inhibited by EGCG, a monooxygenase inhibitor that also inhibits the repulsive action of Semaphorins. It has been shown that H20 2 production by MICAL in cell culture correlates with cell contraction, an activity associated with cytoskeletal reorganization. It was shown that the H20 2 production is a highly regulated process involving interaction of the FAD domain with other MICAL domains and controlled by interaction with the C2 domain of Plexin and with CRMP (collapsin response mediator protein). In addition, the identified interaction of MICAL with CasL has been shown to be an essential component of the regulation of defasciculation, a key early process in axon guidance. The long term goal of this project is to use biophysical methods and atomic resolution 3D structure determination to characterize the regulation of MICAL activity. In this project we concentrate on the interactions among MICAL domains and between MICAL and CasL. Our aims are: 1) to uncover the mechanism by which intramolecular interactions autoinhibit H20 2 production and regulate MICAL redox activity, and 2) to identify and characterize the interactions of MICAL with the SH3 domain of CasL that control defasciculation. Axon guidance signals playa major role in the development of the central nervous system and in nerve regeneration after injury. A detailed characterization of the interactions responsible for these signals is essential for understanding brain development and for the design of pharmacological interventions after nerve injuries.